Research spanning the last decade has helped to elucidate the molecular events attending cell-cell interactions in the body, especially those events involved in the movement and activation of cells in the immune system. See generally, Springer, T., 1990, Nature 346:425-434. Cell surface proteins, and especially the Cellular Adhesion Molecules (“CAMs”) and “Leukointegrins”, including LFA-1, MAC-1 and gp150.95 (referred to in WHO nomenclature as CD18/CD11a, CD18/CD11b, and CD18/CD11c, respectively) have correspondingly been the subject of pharmaceutical research and development having as its goal the intervention in the processes of leukocyte extravasation to sites of injury and leukocyte movement to distinct targets. For example, it is presently believed that prior to the leukocyte extravasation, which is a mandatory component of the inflammatory response, activation of integrins constituitively expressed on leukocytes occurs and is followed by a tight ligand/receptor interaction between integrins (e.g., LFA-1) and one or several distinct intercellular adhesion molecules (ICAMs) designated ICAM-1, ICAM-2, ICAM-3 or ICAM-4 which are expressed on leukocytes. ICAM-1, ICAM-2, and ICAM-3 are expressed on blood vessel endothelial cell surfaces, as well. The interaction of the CAMs with the Leukointegrins is a vital step in the normal functioning of the immune system. Immune processes such as antigen presentation, T-cell mediated cytotoxicity and leukocyte extravasation all require cellular adhesion mediated by ICAMs interacting with the Leukointegrins. See generally, Kishimoto, T. K. and R. R. Rothlein, 1994 Adv. Pharmacol. 25:117≧138.
A group of individuals has been identified which lack the appropriate expression of Leukointegrins, a condition termed “Leukocyte Adhesion Deficiency” (Anderson, D. C. et al., 1985, Fed. Proc, 44:671≧2677 and Anderson, D. C. et al., 1985, J. Infect. Dis., 152:668-689). These individuals are unable to mount a normal inflammatory and/or immune response(s) due to an inability of their cells to adhere to cellular substrates. These data show that immune reactions are mitigated when lymphocytes are unable to adhere in a normal fashion due to the lack of functional adhesion molecules of the CD18 family. By virtue of the fact that LAD patients who lack CD18 cannot mount an inflammatory response, it is believed that antagonism of CD18, CD11/ICAM-1 interactions will also inhibit an inflammatory response.
It has been demonstrated that the antagonism of the interaction between the CAMs and the Leukointegrins can be realized by agents directed against either component. Specifically, blocking of the CAMs, such as for example ICAM-1, or the Leukointegrins, such as for example LFA-1, by antibodies directed against either or both of these molecules effectively inhibits inflammatory responses. In vitro models of inflammation and immune response inhibited by antibodies to CAMs or Leukointegrins include antigen or mitogen-induced lymphocyte proliferation, homotypic aggregation of lymphocytes, T-cell mediated cytolysis and antigen-specific induced tolerance. See generally, Kishimoto, T. K. and R. R. Rothlein, 1994, Adv. Pharmacol. 25:117-138. The relevance of the in vitro studies are supported by in vivo studies with antibodies directed against ICAM-1 or LFA-1. See generally, Rothlein, R. and J. R. Jaeger, 1996, In Therapeutic Immunology (eds) Austen et al. Blackwell Science, Cambridge Mass., p. 347. For example, antibodies directed against LFA-1 can prevent thyroid graft rejection and prolong heart allograft survival in mice (Gorski, A., 1994, Immunology Today 15:251-255). Of greater significance, antibodies directed against ICAM-1 have shown efficacy in vivo as anti-inflammatory agents in human diseases such as renal allograft rejection and rheumatoid arthritis (Rothlein, R. R.; Scharschmidt, L., in: Adhesion Molecules; Wegner, C. D., Ed.; 1994, 1-38, Cosimi, C. B.; et al., J. Immunol. 1990, 144, 4604-4612 and Kavanaugh, A.; et al., Arthritis Rheum. 1994, 37: 992-1004) and antibodies directed against LFA-1 have demonstrated immunosuppressive effects in bone marrow transplantation and in the prevention of early rejection of renal allografts (Fischer, A. et al., Lancet, 1989, 2:1058-1060 and Le Mauff, B.; et al., Transplantation, 1991, 52:291-295).
It has also been demonstrated that a recombinant soluble form of ICAM-1 can act as an inhibitor of the ICAM-1 interaction with LFA-1. Soluble ICAM-1 acts as a direct antagonist of CD18, CD11/ICAM-1 interactions on cells and shows inhibitory activity in in vitro models of immune response such as the human mixed lymphocyte response, cytotoxic T cell responses and T cell proliferation from diabetic patients in response to islet cells (Becker, J. C.; et al., J. Immunol. 1993, 151:7224 and Roep, B. O.; et al., Lancet, 1994, 343:1590).
In addition, several small molecules have been described in the literature which affect the interaction of CAMs and Leukointegrins. A natural product isolated from the root of Trichilia rubra was found to be inhibitory in an in vitro cell binding assay (Musza, L. L.; et al., Tetrahedron, 1994, 50:11369-11378). One series of molecules (Boschelli, D. H.; et al., J. Med. Chem. 1994, 37: 717 and Boschelli, D. H.; et al., J. Med. Chem. 1995, 38: 4597-4614) was found to be orally active in a reverse passive Arthus reaction, an induced model of inflammation that is characterized by neutrophil accumulation (Chang, Y. H.; et al., Eur. J. Pharmacol. 1992, 69: 155-164). Another series of molecules was also found to be orally active in a delayed type hypersensitivity reaction in rats (Sanfilippo, P. J.; et al., J. Med. Chem. 1995, 38: 1057-1059). All of these molecules appear to act nonspecifically, either by inhibiting the transcription of ICAM-1 along with other proteins or act intracellularly to inhibit the activation of the Leukointegrins by an unknown mechanism. None of the molecules directly antagonize the interaction of the CAMs with the Leukointegrins.
Thus, the prior art has demonstrated that molecules which antagonize the binding of the CAMs to the Leukointegrins have therapeutic potential in mitigating inflammatory and immunological responses often associated with the pathogenesis of many autoimmune or inflammatory diseases.
Recently, a small molecule, (R)-5-(4-bromobenzyl)-3-(3,5-dichlorophenyl)-1,5-dimethylimidazolidine-2,4-dione (referred to herein as Compound 1), has been shown to interact specifically with LFA-1 via noncovalent binding to the CD11a chain and prevent LFA-1 from binding to ICAM-1. (Kelly et al., 1999, J. Immunol. 163:5173-5177).
Based on the status of the prior art, there remains a clear need for a method of identifying molecules that specifically bind or modify LFA-1. In addition, there is a need in the clinic for a method for determining LFA-1 receptor occupancy on target cells of a subject treated with such molecules for determining, inter alia: how much compound has reached target cells after treatment; the amount of compound necessary to saturate LFA-1 receptors; and how much receptor occupancy is needed for efficacy.